With over 100 monoclonal antibodies currently being evaluated in clinical study phases 2 or 3, the monoclonal antibody (mAb) market is considered one of the most promising biopharmaceutical markets. Since these drugs have to be delivered to patients in single doses that often exceed 100 mg, there is an urgent need to find suitable formulations that satisfy stability and safety requirements, as well as patient compliance.
Highly concentrated liquid mAb formulations have a higher viscosity than less concentrated formulations, which can hinder their syringeability through more patient-friendly high gauge needles. Furthermore, the tendency of mAb molecules to aggregate exponentially increases with increased concentration, preventing compliance with safety and stability requirements. The delivery of high mAb doses therefore is restricted to large volumes, which generally have to be delivered via infusion. However, this mode of dosing is cost intensive and significantly reduces patient compliance.
For this reason, mAbs in a crystal form are desirable for use as drug substance. However few attempts have been made to evaluate this strategy due to the well known unpredictability associated with crystallization conditions. Although the protein insulin has been successfully crystallized, most other proteins tend to form unordered precipitates rather than crystals. Determining the crystallization conditions for a particular protein is therefore a non-trivial task. To date, there is no general rule that allows one to reliably predict a successful crystallization condition for a protein of choice.
Several screening systems are commercially available (for example, Hampton 1 and 2 and Wizard I and II) that allow, on a microliter scale, screening for potentially suitable crystallization conditions for a specific protein. However, positive results obtained using such screening systems do not necessarily translate into successful crystallization on a larger, industrially applicable batch scale (see Jen, A. et al. (2001) Pharm. Res. 18 (11):1483).
Baldock et al. ((1996) J. Crystal Growth, 168(1-4):170-174) reported on a comparison of microbatch and vapor diffusion for initial screening of crystallization conditions. Six commercially available proteins were screened using a set of crystallization solutions. The screens were performed using a common vapor diffusion method and three variants of a microbatch crystallization method. Out of 58 crystallization conditions identified, 43 (74%) were identified by microbatch, whereas 41 (71%) were identified by vapor diffusion. Twenty-six conditions were identified by both methods, and 17 (29%) would have been missed if microbatch had not been used at all. These data show that the vapor diffusion technique, which is most commonly used in initial crystallization screens, does not guarantee positive results.
Thus, the crystallization of diverse proteins cannot be carried out successfully using defined methods or algorithms. Certainly, there have been technical advances in the last 20-30 years. For example, A. McPherson provides extensive details on tactics, strategies, reagents, and devices for the crystallization of macromolecules. He does not, however, provide a method to ensure that any given macromolecule can indeed be crystallized by a skilled person with a reasonable expectation of success. McPherson states for example: “Whatever the procedure, no effort must be spared in refining and optimizing the parameters of the system, both solvent and solute, to encourage and promote specific bonding interactions between molecules and to stabilize them once they have formed. This latter aspect of the problem generally depends on the specific chemical and physical properties of the particular protein or nucleic acid being crystallized.” (McPherson, A. (1999) Crystallization of Biological Macromolecules. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press, p. 159). It is widely accepted by those skilled in the art of protein crystallization that no one algorithm is reliable for taking a new protein of interest, apply specific process steps, and thereby obtain the desired crystals.
Antibodies are particularly difficult to crystallize, due to the flexibility of the molecule. However, examples of immunoglobulin crystals do exist, such as Bence Jones proteins, which are crystals of an abnormal Ig light chain dimer (Jones, H. B. (1848). Philosophical Transactions of the Royal Society, London, 138:55-62). In addition, crystals of Ig heavy chain oligomer (von Bonsdorf, B., H. Groth, et al. (1938). Folia Haematologia 59:184-208) and human immunoglobulins of normal structure (two heavy chains linked to two light chains) have also been described (Putnam, F. W. (1955) Science 122:275-7; Terry, W. D., et al. (1968) Nature 220(164):239-41; Huber, R., et al. (1976). Nature 264(5585):415-20; Rajan, S. S., et al. (1983) Mol. Immunol. 20(7):787-99; Harris, L. J., et al. (1992) Nature) 360(6402): 369-72, Nisonoff, A., et al. (1968) Cold Spring Harbor Symposia on Quant. Biol. 32:89-93; Connell, G. E., et al. (1973) Canad. J. Biochem. 51(8):1137-41; Mills, L. E., et al. (1983) Annals of Int. Med. 99(5):601-4; and Jentoft, J. E., et al. (1982) Biochem. 21(2):289-294. For example, Margolin and co-workers reported that the therapeutic monoclonal antibody trastuzumab (Herceptin®) could be crystallized (Shenoy, Govardhan et al. 2002) and that crystalline trastuzumab suspensions were therapeutically efficacious in a mouse tumor model, thus demonstrating retention of biological activity by crystalline trastuzumab (Yang, M. X., et al. (2003) Proc. Natl. Acad. Sci. 100(12):6934-6939). However, a predictable and reliable method of forming homogeneous antibody crystal preparations has not been described.
WO-A-02/072636 discloses the crystallization of the whole, intact antibodies Rituximab, Infliximab and Trastuzumab. Most of the crystallization experiments were performed with chemicals that have unclear toxicity, such as imidazole, 2-cyclohexyl-ethanesulfonate (CHES), methylpentanediol, copper sulphate, and 2-morpholino-ethanesulfonate (MES). Many of the examples in this application used seed crystals to initiate crystallization.
Human TNFalpha (hTNFalpha) is considered a causative agent of numerous diseases. There is, therefore, a great need for suitable methods of treating hTNFalpha related disorders. One promising therapeutic approach is the administration of pharmaceutically effective doses of anti-human TNFalpha antibodies. Recently one such antibody, designated D2E7, or generically Adalimumab™, is now on the market under the trade name HUMIRA® (Abbott Laboratories).
WO-A-2004/009776 discloses crystallization experiments on a microliter scale using a sitting drop vapor diffusion technique, which involves mixing equal minute volumes (1 μl) of different crystallization buffers and D2E7 F(ab)′2 or Fab fragments. No methods for the size-controlled crystallization of D2E7 antibody or its fragments were disclosed.
EP-A-0 260 610 discloses the series of murine anti-hTNFalpha monoclonal antibodies, i.e., the neutralizing antibody AM-195, also designated MAK195, as produced by a hybridoma cell line, deposited as ECACC 87050801 with the European Collection of Animal Cell Cultures (ECACC), Health Protection Agency Cultures Collection, Porton Down, Salisbury, United Kingdom on May 8, 1987. An F(ab′)2 fragment of MAK195 (e.g., MAK195F) is also known under the name Afelimomab™. Crystals of MAK195 and of MAK195F are not disclosed. Batch crystallization of these antibodies so far has not been successful.
At present, there is no technical teaching available that provides for the production of anti-hTNFalpha antibody fragment crystals. Moreover, no teaching is available that would provide the size-controlled crystallization of antibody molecules, including antibody fragments, for example, fragments of anti-hTNFalpha antibodies.
A need therefore exists for suitable crystallization conditions, in particular batch crystallization conditions, for antibody and antibody fragments, such as anti-hTNFalpha antibody and antibody fragments, and to establish crystallization process conditions for producing crystal volumes suitable for industrial production. A need also exists for a crystallization process that does not make use of toxic agents, which might negatively affect the pharmaceutical applicability of such antibodies. Still another need exists for a crystallization method for antibodies or antibody fragments, such as Fab or F(ab′)2 fragments, that allows for the selection and control of crystal size.